Portal Vein Islet Isografts Resemble Preneoplastic ... - Europe PMC

Amenican Journal of Pathology, Vol. 148, No. 4, April 1996

Copyright © American Societyfor Investigative Pathology

Altered Liver Acini Induced in Diabetic Rats by Portal Vein Islet Isografts Resemble Preneoplastic Hepatic Foci in Their Enzymic Pattern

Frank Dombrowski,* Erika Filsinger,t Peter Bannasch,t and Ulrich Pfeifer* From the Pathologisches Institut,* Universitat Bonn, Bonn, Germany, and Abteilung far Cytopathologie,t Deutsches Krebsforschungszentrum, Heidelberg, Germany

As demonstrated previously, liver acini draining the blood from intraportally transplanted pancreatic islets in streptozotocin-diabetic rats are altered in various respects. The hepatocytes in these acini store glycogen and/or fat, and they show an increase in proliferation as weU as in apoptotic activity. Thus, they are phenotypicaly similar to carcinogen-induced preneoplastic liver foci (glycogen-storing foci and sometimes also mixed ceU foci). By means of catalytic enzyme histochemistry or immunohistochemistry, we investigated the activity of key enzymes of alternative pathways of carbobydrate metabolism and some additional marker enzymes (weU known from studies on preneoplastic hepatic foci) in the altered liver acini surrounding the islet isografts. In addition, the expression ofglucose transporter proteins 1 and 2 (GLUT-1 and GLUT-2) were investigated immunohistochemically. The activities of hexokinase, pyruvate kinase, glyceraldebyde-3-phosphate debydrogenase, and glucose-6-phosphate debydrogenase were increased, whereas the activities of glycogenphosphorylase, adenylate cyclase, glucose-6phosphatase, and membrane-bound adenosine triphosphatase were decreased in the altered liver acini. The expression of GLUT-2 was also decreased. GLUT-1 and glutathione S-transferase placentalform were not expressed, and the activities of glycogen synthase and y-glutamyltransferase remained unchanged. AU changes of the enzyme activities were in line with the weU known effects of insulin and resembled alter-

ations characteristic of preneoplastic liver foci observed in different models of hepatocarcinogenesis. It remains to be clarified in long-term experiments whether or not these foci represent preneoplastic lesions and may proceed to neoplasia. (Am J Pathol 1996, 14&1249 -1256)

After the transplantation of isolated isogenous islets of Langerhans via the portal veins into the livers of streptozotocin-diabetic rats, the animals are supplied with insulin from the transplanted islets.1-7 If the number of islets transplanted is too low to compensate completely the diabetic state, the transplanted islets are chronically in a highly stimulated state due to the persisting hyperglycemia.7 Insulin is known to have growth-promoting effects in many short-term experiments,8-15 but the consequences of a long-term local hyperinsulinism in the liver in vivo are poorly understood.7'16'17 Hepatocellular alterations confined to the acini draining the blood from the grafted islets have been described recently in a model of low-number-islet transplantation7 as follows. The hepatocytes accumulate glycogen, and often also fat, in excess. The border between the altered acini and the surrounding liver parenchyma is well defined. The altered acini are in a hyperproliferative state, and the number of apoptotic bodies is considerably increased. The focal character, the morphology, and the alterations in glycogen content of the altered liver acini are similar to those in early preneoplastic liver lesions, namely in glycogen-storing foci and mixed cell foci.18'19 With respect to the increased proliferation and apoptotic elimination of the hepatocytes, the altered liver acini likewise resemble preneoplastic Accepted for publication December 18, 1995. Address reprint requests to Dr. Frank Dombrowski, Pathologisches Institut, der Universitat Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany.

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liver lesions.2021 The question of whether or not apoptosis is counterbalancing cell replication in preneoplasia under all experimental conditions remains a matter of debate.21'22 Characteristic enzymatic alterations were observed in preneoplastic liver foci emerging in several models of hepatocarcinogenesis.23 25 We examined, therefore, whether the altered liver acini appearing rapidly after islet transplantation are characterized by changes in enzyme activities similar to those observed in preneoplastic liver foci.

Materials and Methods Animals and Tissue Sampling Highly inbred male Lewis rats (Experimental Animal Center, Hannover, Germany), weighing 250 to 300 g at the beginning of the experiment, were made diabetic by a single intravenous dose of streptozotocin (80 mg/kg body weight). Diabetes was defined by a nonfasting blood glucose level greater than 400 mg/ dl, which was reached 1 to 3 days after streptozotocin injection. The body weight and capillary blood glucose (tip of the tail) were measured daily (HaemoGlucotest 1-44 R and Reflolux 11, BoehringerMannheim, Mannheim, Germany). On the 2nd day after becoming diabetic, an insulin therapy as described elsewere21 was started and continued until the day of transplantation. The treatment with insulin was performed to avoid greater weight loss and to have a normoglycemia at the time of the transplantation. Isologous islet transplantation of approximately 450 islets via the portal vein was carried out on 14 animals (for details of the transplantation procedure, see Ref. 7). Animals were killed 2 to 21 days after transplantation. All experiments were conducted according to National Institutes of Health guidelines for animal care and experimentation.

Histochemistry of Enzymes and Metabolic Products Pieces from frozen liver tissue of four rats were frozen onto the same tissue holder, and serial sections of all pieces were cut simultaneously in a cryostat (Jung, Nussloch, Germany).27 The sections were mounted onto the same slide or the same membrane and incubated according to the respective histochemical reaction. With this technique, it was possible not only to produce sections of the same thickness but also to treat them simultaneously under

identical conditions for the specific histochemical assays. The thickness of the sections was 14 ,um for glycogen synthetase (SYN) and glycogen phosphorylase (PHO) and 6 ,um for all other enzymes and for immunohistochemistry. The majority of enzymes studied catalyze important reactions in different pathways of energy metabolism, particularly in the alternative pathways of carbohydrate metabolism (glycogen synthesis and breakdown, glycolysis, and the pentose phosphate pathway), as demonstrated by procedures described in detail elsewhere, and include SYN and PHO,28 adenylate cyclase (ADC),29 glucose-6-phosphatase (G6Pase),3° glucose-6phosphate dehydrogenase (G6PDH),31 glyceraldehyde-3-phosphate dehydrogenase (GAPDH),32 pyruvate kinase,33 and hexokinase (HK).34 Expression of the two important glucose transporter proteins GLUT-2 (which is the typical glucose transporter in hepatocytes of adult liver) and the erythroid/ brain-type glucose transporter GLUT-1 (representing the typical glucose transporter in fetal liver parenchyma) were studied immunohistochemically by the alkaline phosphatase anti-alkaline phosphatase technique.35 Expression of glutathione Stransferase placental form was investigated immunohistochemically according to Sato et al.36 In addition, membrane-bound ATPase and y-glutamyltransferase (GGT),37 frequently used marker enzymes for foci of altered hepatocytes in rat,23 were investigated. Furthermore, serial cryostat sections were stained for basophilia with toluidine blue, for the presence of neutral lipids with Fettrot B, and for the presence of glycogen with the periodic acidSchiff reaction (PAS).

Evaluation of Histochemical Reactions The intensity of the histochemical parameters in the serial sections was estimated semiquantitatively using five grades (no change, increase, strong increase, decrease, and strong decrease) as compared with the reaction in the unaltered tissue of the same specimen (internal control).

Results Histology With the PAS reaction it was easy to identify foci of altered liver parenchyma surrounding the transplanted islets of Langerhans (Figure 1) at every stage of the experiment (day 2 to day 21 after the transplantation). In these foci the hepatocytes were much larger than in the unaltered parenchyma and

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Figure 1. PAS reaction shows glycogen storage of altered liver acini surrounding two pancreatic islets (*) 11 days after transplantation. 7Te topography of the central veins (arrowheads) prove the interpretation that the focal changes represent acini. Cryostat section. Magnification, X 100.

most of them stored large quantities of PAS-positive material that had been identified earlier as glycogen by electron microscopy.5'7 Some of the foci were composed of a mixture of glycogen-rich and glycogen-poor hepatocytes (mixed cell foci). The basophilic cells often contained mitotic figures. In addition, apoptotic bodies were frequently found. These alterations have been reported in detail and quantified previously7 and were not calculated again in this study. Foci of predominantly glycogen-depleted hepatocytes and liver tumors were not found. The foci of altered hepatocytes were regularly arranged as polygons bordering on central veins or collecting veins. The transplanted islets were always located in portal triads closely related to these foci (Figure 1). It is evident from this typical architecture of the foci that they correspond anatomically to liver acini. The size of the lesions depends on the number of the acini involved.

Enzyme Histochemistry In the unaltered liver acini the histochemical pattern of the liver parenchyma reflected the well known

metabolic zonation showing gradients in enzyme activities from zone 1 to zone 3 of the acini, or vice versa. Thus, the reaction of the histochemical assay for G6Pase, G6PDH, GAPDH, PHO, and SYN was stronger in zone 1 than in zone 3, whereas those for ADC, pyruvate kinase, and HK were more pronounced in zone 3. In hepatocytes the reaction product of membrane-bound ATPase was limited to the plasma membrane surrounding the bile canaliculi and was also positive in bile ducts and blood vessels. GGT was positive in bile ducts and some bile canaliculi of hepatocytes of zone 1. The histochemical patterns of the altered liver acini are summarized in Table 1 and some of them are shown in Figure 2. The majority of altered acini were characterized by increased activities of G6PDH, HK (Figure 2, c and d), and GAPDH. The activities of PHO, ADC, and G6Pase were often reduced (Figure 2, e-g). The merlbrane-bound ATPase was either slightly decreased (Figure 2h) or slightly increased. SYN was often unchanged. GGT was seen orthotopically only in bile ducts and sometimes in bile canaliculi of hepatocytes of zone 1 but was not overexpressed in any case.

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The increased activities of the glycolytic enzymes HK, GAPDH, and pyruvate kinase, and the decreased activity of the gluconeogenic enzyme G6Pase are well known effects of insulin that correlate with the hyperglycemia in the diabetic animals and provide evidence for the postulated hyperinsulinism in the altered acini.7 The strongly increased activity of G6PDH of the altered liver acini after islet transplantation shows the most marked contrast to the unaltered acini, in which the G6PDH activity is very low or absent. By the histochemical demonstration of this enzyme, altered acini can be detected simply with the naked eye. The hyperproliferative nature of the altered acini has been demonstrated

Table 1. Histochemical Pattern of Altered Liver Acini after Low-Number Intraportal Islet Transplantation in Diabetic Rats

Intensity NC( T)

Basophilia Glycogen Fat PHO SYN ADC mATPase G6Pase HK GAPDH PK G6PDH GGT GST-P GLUT-1 GLUT-2

T T( T) NC(I T)

I (NC) T (NC) 1(T T) NC NC NC

The intensity of the histochemical parameters in focal lesions estimated semiquantitatively in comparison to the unaltered liver acini of the same tissue sections, using five grades: NC, no change; increase; T T, strong increase; decrease; and strong decrease. When different intensities were observed, the less frequent alteration is given in brackets. mATPase, membrane-bound ATPase; PK, pyruvate kinase; GST-P, glutathione, S-transferase placental form. was

,

4,

4,

Immunohistochemistry GLUT-2 was expressed at the cell membranes of normal hepatocytes but was decreased in the altered acini (Figure 2i). GLUT-1 was positive at the cell membranes of perivenular hepatocytes, bile ducts, and single-neutrophil granulocytes and lymphocytes in the blood vessels. There was no expression of GLUT-1 and glutathione S-transferase placental form in the liver parenchyma of the altered acini (Table 1).

Discussion The morphological findings agree with our results reported previously for the same animal model.7 It is evident that the morphological alterations are restricted to anatomically defined liver acini. The enzyme histochemical results of this study show that the focal morphological alterations have a background in focal functional enzymatic alterations; the decreased activity of PHO accompanied by a decreased activity of ADC, which is down-regulated by insulin, may be causally related to the excessive storage of glycogen. Surprisingly, SYN was not changed. In the presence of hyperglycemia, this finding is difficult to understand, but it is in accordance with observations in preneoplastic hepatic glycogen storage foci.38

previously.7 The focal excessive storage of glycogen (glycogenosis) and often also fat in the altered liver acini is reminiscent of glycogen-storing foci and mixed cell foci induced by different carcinogenic agents,18'19'23 which have been considered to elicit insulin-like effects in the hepatocytes.39 In this context, it is interesting to note that in preneoplastic hepatic foci an overexpression of the protein kinase Raf-1 has been demonstrated by in situ hybridization.40 Raf-1 is involved in the signal transduction pathway stimulated by insulin,41 which has previously been shown to activate other SerlThr protein kinases such as Ras and mitogen-activated protein kinase (for literature see Ref. 41). The focal glycogenosis in preneoplastic hepatic foci has been shown to be associated with a number of enzymatic changes. 23-25,38,39,42 Thus, a reduction in the activities of ADC, PHO, and frequently also G6Pase and a-glucosidase in preneoplastic hepatic foci suggest an early disturbance of the phosphorolytic and hydrolytic glycogen breakdown. The resulting glycogenosis may persist for weeks and months but is eventually replaced by a gradual reduction in the glycogen. This metabolic shift is associated with the activation of enzymes in alternative pathways of carbohydrate metabolism such as the pentose phosphate pathway and glycolysis, which have been known to be increased in hepatocellular neoplasms for a long time.19 Overexpression of G6PDH is one of the most striking enzymatic changes in preneoplastic hepatic foci,39,43-46 indicating an elevated activity of the pentose phosphate pathway. In addition to its other functions, the pentose phosphate pathway produces precursors for DNA and RNA synthesis. Baba et a147 have shown by autoradiographic studies in foci of altered hepatocytes negative and positive for G6PDH that the increased activity of this enzyme is closely related to proliferation of the preneoplastic and neoplastic cells in the liver. It is, therefore, con-

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v.,

.i:.. :...

Fiaure ,zortirm-c /j" alterea tiltor'O.-I i ver acinus 2i "?l aays '4--, t" 1-1. Sprial auffttw uunf ar .I.w-_2. A to ajter islet transplantation. Glycogen storage is sbown by PAS (A) . Altered hepatocytes also often store fat (B) . Activities ofpentose phosphate pathway key enzyme G6PDH (C) and enzymes of glycolysisreaction such as HK (D) are

increased, whereas activities of glycogen phophorylase (E), G6Pase (F), ADC (G), and membrane-bound ATPase (H) were The morphological aspect of this altered liver acinus, the pattern of altered enzymatic activities, and the reduced expression of GLUT-2 (1)decreased. resemble those ofpreneoplastic liver lesions. Magnification, X34.

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ceivable that the focal metabolic changes gradually developing during hepatocarcinogenensis provide a growth advantage eventually resulting in neoplastic transformation. The hypothesis that the molecular changes underlying the aberrations in carbohydrate metabolism that emerge during hepatocarcinogenesis are causally related to neoplastic transformation is supported by the ever increasing number of case reports on patients suffering from inborn hepatic glycogenosis type (von Gierke's disease), most of whom develop hepatic adenomas and carcinomas when they pass through adolescence.4248 Although there are many obvious similarities between the altered liver acini appearing after islet transplantation and preneoplastic liver foci, some differences have to be taken into account: In contrast to the preneoplastic hepatic foci, the emergence of which usually requires a lag period of several weeks,23 the alterations in the liver acini produced by the portal vein islet isografts develop within

days.5'7 Whereas the size of foci of altered hepatocytes induced by chemical carcinogens expands slowly during the preneoplastic phase, the islet-induced focal lesions occupy the majority of hepatocytes in pre-existing acini from the very beginning. Thus, these focal lesions cannot be assumed to be of monoclonal origin, as has been discussed for preneoplastic lesions by several authors49-51 but has been questioned by others.52 The alterations in focal lesions produced by pancreatic islet transplantation appear to depend on a sustained local increase in concentration of islet hormones, especially of insulin. This has been concluded from the observation that focal alterations vanish or do not appear when the hyperglycemia ceases after transplantation of large amounts of pancreatic islet tissue.5'7 Only when a persisting hyperglycemia stimulates insulin secretion in the transplanted islets, focal lesions develop.7 Accordingly, altered liver acini were not observed after islet transplantation into the livers of nondiabetic rats.7 Under many experimental conditions, preneoplastic foci have been shown to persist after withdrawal of the inducing carcinogenic agent for weeks and months. Only under certain experimental conditions, particularly after administration of a high sublethal dose of carcinogens, has a partial reversion of focal lesions been reported, the mechanism of which is poorly understood 23,53 The complete lack of an expression of GGT and glutathione S-transferase placental form in altered liver acini appearing after islet transplantation represents an additional discrepancy between these le-

sions and the majority of foci of altered hepatocytes produced by established hepatocarcinogens.36'54 However, specific types of preneoplastic foci of altered hepatocytes, such as tigroid cell foci and amphophilic cell foci, also never express GGT.55'56 The development of hepatic neoplasia after islet transplantation via the portal vein has not been reported. However, the consequences of long-term systemic hyperglycemia and local hyperinsulinemia in the liver after transplantation of a low number of islets of Langerhans into the liver have never been investigated. From the results obtained in this study it would appear that additional investigations into long-term experiments should take place to clarify whether the altered acini induced by islet transplantation persist for a long time and may eventually give rise to hepatocellular neoplasms. A detailed analysis of this experimental system might be of considerable help in our understanding of metabolic aberrations, which have been known for some time to occur in preneoplastic lesions and seem to be closely linked to the process of the neoplastic conversion of hepatocytes.

Acknowledgments The skillful technical assistance of Gisela Beckhaus, Jbrg Bedorf, Holger Grage, Mathilde Hau-Liersch, and Inge Heim and the excellent photographic work of Gerrit Klemm and Bjbrn Bieser are greatfully acknowledged. The authors thank Dr. Hans-Jorg Hacker for many helpful discussions, comments, and interpretations.

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Lacy PE: A morphologic study of intrahepatic portalvein islet isografts. Diabetes 1977, 26:201-214 6. Bretzel RG: Inseltransplantation und Diabetes mellitus. Experimentelle Grundlagen und klinische Versuche. MOnchen, Richard Pflaum Verlag, 1984 7. Dombrowski F, Lehringer-Polzin M, Pfeifer U: Hyperproliferative liver acini after intraportal islet transplantation in streptozotocin-induced diabetic rats. Lab Invest 1994, 71 :688-699 8. Francavilla A, Starzl TE, Porter K, Foglieni CS, Michalopoulos GK, Carrieri G, Trejo J, Azzarone A, Barone M, Zeng QH: Screening for candidate hepatic growth factors by selective portal infusion after canine Eck's fistula. Hepatology 1991, 14:665-670 9. Ricordi C, Flye MW, Lacy PE: Renal subcapsular transplantation of clusters of hepatocytes in conjunction with pancreatic islets. Transplantation 1988, 45:1148-1151 10. Starzl TE, Porter KA, Kashiwagi N, Putnam CW: Portal hepatotrophic factors, diabetes mellitus and acute liver atrophy, hypertrophy and regeneration. Surg Gynecol Obstet 1975, 141 :843-858 11. Starzl TE, Watanbe K, Porter KA, Putnam CW: Effects of insulin, glucagon, and insulin/glucagon infusions on liver morphology and cell division after complete portocaval shunt in dogs. Lancet 1976, 1:821-825 12. Bucher NLR, Swaffield MN: Regulation of hepatic regeneration in rats by synergistic action of insulin and glucagon. Proc Natl Acad Sci USA 1975, 72:11571160 13. Pfeifer U: Application of test substances to the surface of rat liver in situ: opposite effects of insulin and isoproterenol on cellular autophagy. Lab Invest 1984, 50: 348-354 14. Pfeifer U: Inhibition by insulin of the formation of autophagic vacuoles in rat liver: a morphometric approach to the kinetics of intracellular degradation by autophagy. J Cell Biol 1978, 78:152-167 15. Reed GB, Grisham JW: Insulin and hydrocortisone effects on viability and glycogen stores of postnatal rat liver organ culture. Lab Invest 1975, 33:298-304 16. Wanless IR, Bargman JM, Oreopoulos DG, Vas SI: Subcapsular steatonecrosis in response to peritoneal insulin delivery: a clue to the pathogenesis of steatonecrosis in obesity. Modern Pathol 1989, 2:69-74 17. Burrows CJ, Jones AW: Hepatic subcapsular steatosis in a patient with insulin dependent diabetes receiving dialysis. J Clin Pathol 1994, 47:274-275 18. Bannasch P: The cytoplasm of hepatocytes during carcinogenesis: electron and light microscopical investigation after nitrosomorpholine-intoxicated rat liver. Recent Res Cancer Res 1968, 19:1-100 19. Bannasch P, Mayer D, Hacker HJ: Hepatocellular glycogenosis and hepatocarcinogenesis. Biochim Biophys Acta 1980, 605:217-145 20. Columbano A, Ledda-Columbano GM, Rao PM, Rajalakshmi S, Sarma DSR: Occurrence of cell death (apoptosis) in preneoplastic and neoplastic liver cells. Am J Pathol 1984, 116:441-446

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